Recently there has been an increased interest in predictive, preventative, and particularly personalized medicine which requires diagnostic tests with higher fidelity, e.g., sensitivity and specificity. Multiplexed measurement platforms, e.g., protein arrays currently used in research, are among the promising diagnostics technologies for the near future. The samples in these tests can be human body fluids such as blood, serum, saliva, biological cells, urine, or other biomolecules but can also be consumables such as milk, baby food, or water. Within this field there is a growing need for low-cost, multiplexed tests for biomolecules such as nucleic acids, proteins, and also small molecules. Achieving the sensitivity and specificity needed in such tests is not without difficult challenges. Combining these tests with integrated electronics and using CMOS technology has provided solutions to some of the challenges.
The two main limitations in a detection assay include sensitivity and cross-reactivity. Both of these factors affect the minimum detectable concentration and therefore the diagnostic error rate. The sensitivity in such tests is generally limited by label detection accuracy, association factor of the probe-analyte pair (for example an antibody-antigen pair), and the effective density of probe molecule (for example probe antibody) on the surface. Other molecules in the biological sample can also affect the minimum detectable concentration by binding to the probe molecule (for example the primary antibody), or by physisorption of the analyte to the surface at the test site. The detection agent (for example a secondary antibody) may also physisorb to the surface causing an increase in the background signal. Solving the cross-reactivity and background problem can take a significant amount of time in the assay development of a new test and increases the cost and complexity of the overall test. The assay is typically optimized by finding the best reagents and conditions and also by manufacturing the most specific probe molecule (for example antibody). This results in a long development time, the infeasibility of tests in some cases, and a higher manufacturing cost. For example a typical development of an ELISA assay requires several scientists working for more than a year finding the correct antibody as part of the assay development. Cross-reactivity of the proteins may be the source of the failure of such an effort.
A biosensor providing a multiple site testing platform was thought to provide a solution to some of the above described limitations in assay development. US Published Patent Application US 2011/0091870 describes such biosensor having multiple sites that could be subjected to different reaction conditions to modulate the binding of the biomolecular analyte (for example proteins) to the probe molecule. For example the signal detected in a biosensor having four sites also can have several components, e.g. four. These four terms may correspond to the concentration of the biomarker of interest, concentration of interfering analytes in the sample that bind non-specifically to primary antibody (probe molecule) sites and prevent the biomarker to bind, concentration of interfering analytes in the sample that form a sandwich and produce wrong signal, and finally the concentration of interfering analytes in the sample that physisorb to the surface and produce wrong signal. Each term is also proportional to a binding efficiency factor, αij, which is a function of the molecule affinities and other assay conditions, e.g., mass transport. By controlling the condition at each site separately, different sites will have different efficiency factors. Accurate measurement of the signal at each site will result in multiple equations and multiple unknowns for example,
  {                                                        S              1                        =                                                            α                  11                                ⁢                                  C                  an                                            -                                                α                  12                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  13                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  14                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              2                        =                                                            α                  21                                ⁢                                  C                  an                                            -                                                α                  22                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  23                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  24                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              3                        =                                                            α                  31                                ⁢                                  C                  an                                            -                                                α                  32                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  33                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  34                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              4                        =                                                            α                  41                                ⁢                                  C                  an                                            -                                                α                  42                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  43                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  44                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                            ⁢        ⁢          C      an      where Can corresponds to the targeted biomolecular analyte concentration and Cj1, Cj2, Cj3 correspond to the total concentration of molecules which result in different terms in background signal.
Accurate and precise control of the assay conditions at different sites to generate large changes in the binding efficiency factors is important in the performance of such biosensor as a detection system for a biomolecular analyte of interest. In co-pending U.S. application Ser. No. 13/543,300 (the content of which is incorporated herein by reference in its entirety) such biosensors and such methods are described that can be readily integrated with a CMOS, electrode array, or TFT based setup to generate large change in binding efficiencies between test sites in a biosensor having an array of multiple test sites. In order to accurately measure the biomolecular analyte of interest the biosensor requires a high degree of reliability and reproducibility. Variations in the modulation of the local pH due to repeated use of the biosensor and variations between subsequent measurements may decrease the accuracy of the determination of the biomolecular analyte of interest by such biosensor. As such the modulation of the pH at each site of the multisite array of the biosensor needs to be accurately controlled and variations in such pH modulation need to be corrected. Therefore, there is a need for a biosensor in which the pH can be accurately, reliably, and reproducibly controlled at each of the multisite array test sites.